Modulation of Kinase Activity and Oncogenic Properties by Alternative Splicing Reveals a Novel Regulatory Mechanism for B-Raf

Abstract

Members of the raf oncogene family encode serine/ threonine protein kinases, which activate the mitogenactivated protein kinase kinase MEKs (MAPK or ERK kinases) through direct interaction and phosphorylation. Several recent studies have revealed interesting differences between two members of this family, Raf-1 and B-Raf, regarding their activation, regulation, and kinase activity. In particular, B-Raf was shown to display higher MEK kinase activity than Raf-1. By using both two-hybrid analysis and coimmunoprecipitation experiments, we demonstrate here that B-Raf also markedly differs from Raf-1 by a higher affinity for MEK. We previously reported that the B-raf gene encodes multiple protein isoforms resulting from complex alternative splicing of two exons (exons 8b and 10) located upstream of B-Raf kinase domain. In the present study, we show that these naturally occurring modifications within the protein sequence markedly modulate both the biochemical and oncogenic properties of B-Raf. The presence of exon 10 sequences enhances the affinity for MEK, the basal kinase activity, as well as the mitogenic and transforming properties of full-length B-Raf, whereas the presence of exon 8b sequences seems to have opposite effects. Therefore, alternative splicing represents a novel regulatory mechanism for a protein of the Raf family.

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

“…However, the B-Raf Nn/V600E mutant displayed comparable biological activity in HEK293 (ERK phosphorylation), PC12 (differentiation) and MCF-10A cells (ERK phosphorylation, transformation and EMT induction) as B-Raf V600E (Figures 1, 5-8). Similarly, while our Presumably, this open conformation of B-Raf that is achieved prior to interaction with Ras-GTP also explains why it binds MEK-1 with higher affinity than Raf-1 (Papin et al, 1998). Induction of Ras-GTP by extracellular signals results in the recruitment of pre-activated B-Raf to the plasma membrane where it is fully activated by activation segment kinases.…”

“…Induction of Ras-GTP by extracellular signals results in the recruitment of pre-activated B-Raf to the plasma membrane where it is fully activated by activation segment kinases. This assumption is supported by the observation that a membrane-targeted B-Raf-CAAX protein displays constitutive MAPKKK and high transforming activities (Papin et al, 1998;Carey et al, 2003). Phosphorylation of the activation segment destabilizes the inactive conformation of the kinase domain (Wan et al, 2004) and renders B-Raf fully active.…”

“…The Raf-kinase family comprises the A-Raf, B-Raf and Raf-1 isoforms in vertebrates as well as D-Raf and LIN-45 in Drosophila and Caenorhabditis, respectively. B-Raf, the major ERK activator in vertebrates, is required for the maintenance of basal ERK activity and displays the most potent transforming activity (Papin et al, 1998;Brummer et al, 2002;Mercer and Pritchard, 2003). All isoforms share three highly conserved regions (CRs; Figure 1a): the N-terminal CR1 contains the Ras-guanine 5 0 -triphosphate (GTP)-binding domain (RBD), which initiates the interaction with Ras-GTP through a conserved arginine residue (R188 in B-Raf) that is required for the recruitment and activation of Raf at the plasma membrane.…”

Interferon-α 2b reduces phosphorylation and activity of MEK and ERK through a Ras/Raf-independent mechanism

“…Once activated by autophosphorylation or upstream kinases (Gomez and Cohen, 1991;Dent et al, 1992;Howe et al, 1992;Kyriakis et al, 1992;Ahn et al, 1993;Itoh et al, 1993;Matsuda et al, 1993;Posada et al, 1993;Gardner et al, 1994;Haystead et al, 1994;Papin et al, 1998), MEK activity is the main upstream mechanism leading to phosphorylation of both tyrosine and serine/ threonine residues and subsequent activation of ERK (Seger and Krebs, 1995). This, in turn, results in the regulation of a large number of proteins both in the cytoplasm and, following ERK translocation, in the nucleus (Khokhlatchev et al, 1998).…”

Régulation de la survie cellulaire par les kinases de la famille RAF

“…Les isoformes de RAF diffèrent entre elles en termes d'activité kinase. Toutes trois sont capables d'activer une unique cible, MEK1/2 (voir Glossaire), mais présentent d'importantes différences d'activité vis-à-vis de ce substrat [2][3][4][5][6]. Globalement, B-RAF est 1 Voir le glossaire.…”